It is proposed that neoplastic cells differ from normal cells in several aspects of DNA synthesis. A long-range goal is to determine which aspects of DNA replication are altered in transformed cells in culture as compared with normal cells. DNA is not a static structure but undergoes continuous structural alterations, detected as breaks and single-strand nuclease-sensitive sites, as cells proceed through the cell cycle. DNA will be isolated from transformed fibroblasts and mutants temperature-sensitive for the transformed phenotype and examined for structural alterations in parental and replicative DNA of five subcompartments of the S-phase. Parental DNA of several subcompartments of G1 also will be examined. G2 DNA will be examined as a control. The interconversion of replicative intermediates, which differs in transformed and normal fibroblasts, will be examined in ts mutant cells at the permissive and nonpermissive temperatures. In contrast to frequent assumptions, the rate of DNA synthesis is not constant during the S-phase but fluctuates such that relative values of 30%, 60%, and 100% can occur. The fastest rates for HeLa and 2RA cells are at the beginning and end of S-phase, whereas WI-38 cells exhibit a gradual increase in rates from beginning to end of S-phase. It is proposed that single-stranded sites on DNA and the activities of DNA polymerases alpha, beta, and gamma of the single-strand specific chromatin-bound DNA polymerase and of DNA ligase are possible candidates for the factors that determine the observed variations in the rates of DNA synthesis as cells proceed through S. The measured enzyme activities will be correlated with quantitative rates of DNA synthesis determined with a flow cytometer in five S-phase compartments. (I)